2. Mice were injected with two strains of pneumococcus: an encapsulated (S) strain and a non-encapsulated (R) strain.

a. The S strain is virulent (the mice died); it has a mucous capsule and forms “shiny” colonies.

b. The R strain is not virulent (the mice lived); it has no capsule and forms “dull” colonies.

3. In an effort to determine if the capsule alone was responsible for the virulence of the S strain, he injected mice with heat-killed S strain bacteria; the mice lived.

4. Finally, he injected mice with a mixture of heat-killed S strain and live R strain bacteria.

a. The mice died; living S strain pneumococcus was recovered from their bodies.

b. Griffith concluded that some substance necessary for synthesis of the capsule—and therefore for virulence—must pass from dead S strain bacteria to living R strain bacteria so the R strain were transformed.

c. This change in phenotype of the R strain must be due to a change in the bacterial genotype, suggesting that the transforming substance passed from S strain to R strain.

1. Transformation experiments today are important especially in biotechnology labs.

2. Transformation of organisms is being used in commercial products.

3. In order to illustrate that transferring genes was possible from one organism to another, scientists used a green fluorescent protein from jellyfish and transferred it to other organisms. The result was that these organisms glowed in the dark.

4. Mammalian genes have the ability to function in other species: bacteria, invertebrates, plants.

D. The Structure of DNA

1. Erwin Chargaff (1940s) analyzed the base content of DNA.

2. It was known DNA contained four different nucleotides:

a. Two with purine bases, adenine (A) and guanine (G); a purine is a type of nitrogen-containing base having a double-ring structure.

b. Two with pyrimidine bases, thymine (T) and cytosine (C); a pyrimidine is a type of nitrogen-containing base having a single-ring structure.

3. Results: DNA does have the variability necessary for the genetic material.

4. For a species, DNA has the constancy required of genetic material.

5. This constancy is given in Chargaff’s rules:

a. The amount of A, T, G, and C in DNA varies from species to species.

b. In each species, the amount of A = T and the amount of G = C (A +G = T +C).

6. The tetranucleotide hypothesis (proposing DNA was repeating units of one of four bases) was disproved: each species has its own constant base composition.

7. The variability is in base sequences is staggering; a human chromosome contains about 140 million base pairs.

8. Since any of the four possible nucleotides can be present at each nucleotide position, the total number of possible nucleotide sequences is 4140 x 106 = 4140,000,000.

9. Rosalind Franklin produced X-ray diffraction photographs.

10. Franklin’s work provided evidence that DNA had the following features:

a. DNA is a helix.

b. Some portion of the helix is repeated.

11. American James Watson joined with Francis H. C. Crick in England to work on the structure of DNA.

12. Watson and Crick received the Nobel Prize in 1962 for their model of DNA.

13. Using information generated by Chargaff and Franklin, Watson and Crick constructed a model of DNA as a double helix with sugar-phosphate groups on the outside, and paired bases on the inside.

14. Their model was consistent with both Chargaff’s rules and Franklin’s X-ray diffraction studies.

15. Complementary base pairing is the paired relationship between

purines and pyrimidines in DNA: A is hydrogen-bonded to T and G is hydrogen-bonded to C.

12.2 Replication of DNA

1. DNA replication is the process of copying a DNA molecule. Replication is semiconservative, with each strand of the original double helix (parental molecule) serving as a template (mold or model) for a new strand in a daughter molecule. This process consists of:

a. Unwinding: old strands of the parent DNA molecule are unwound as weak hydrogen bonds between the paired bases are “unzipped” and broken by the enzyme helicase.

b. Complementary base pairing: free nucleotides present in the nucleus bind with complementary bases on unzipped portions of the two strands of DNA; this process is catalyzed by DNA polymerase.

c. Joining: complementary nucleotides bond to each other to form new strands; each daughter DNA molecule contains an old strand and a new strand; this process is also catalyzed by DNA polymerase.

A. Aspects of DNA Replication (Biological Systems reading)

1. For complementary base pairing to occur, the DNA strands need to be antiparallel, as discovered by Watson and Crick.

2. One strand of DNA is 5’ at the top and the other strand is 3’ at the top of the strand.

3. During replication the DNA polymerase can only join to the free 3’ end of the previous nucleotide.

4. DNA polymerase cannot start the synthesis of a DNA chain, so an RNA polymerase lays out an RNA primer that is complementary to the replicated strand.

5. Now the DNA polymerase can join the DNA nucleotides to the 3’ end of the new strand.

6. The helicase enzyme unwinds the DNA and one strand (called the leading new strand) can be copied in the direction of the replication fork.

7. The other strand of DNA is copied in the direction away from the fork, and replication begins again.

a. This new lagging strand is discontinuous and each segment is called an Okazaki fragment, after the scientist who discovered them.

8. Replication is only complete when RNA primers are removed.

9. During replication, DNA molecules gets smaller and smaller.

10. The end of eukaryotic DNA molecules have nucleotide sequences called telomeres.

a. Telomeres don’t code for proteins. They are repeats of short nucleotide sequences (i.e., TTAGGG).

11. Normal mammalian cells divide approximately 50 times and then stop. However in cancer cells the telomerase can be turned on and cancer cells then divide without limit.

B. Prokaryotic Versus Eukaryotic Replication

1. Prokaryotic DNA Replication

a. Bacteria have a single loop of DNA that must replicate before the cell divides.

b. Replication in prokaryotes may be bidirectional from one point of origin or in only one direction.

c. Replication only proceeds in one direction, from 5' to 3'.

d. Replication begins at a special site on a bacterial chromosome, called the origin of replication.

Four nucleotides based on 3-unit codons allows up to 64 different amino acids to the specified.

Finding the Genetic Code

a. Marshall Nirenberg and J. Heinrich Matthei (1961) found that an enzyme that could be used to construct synthetic RNA in a cell-free system; they showed the codon UUU coded for phenylalanine.

b. By translating just three nucleotides at a time, they assigned an amino acid to each of the RNA codons, and discovered important properties of the genetic code.

c. The code is degenerate: there are 64 triplets to code for 20 naturally occurring amino acids; this protects against potentially harmful mutations.

d. The genetic code is unambiguous; each triplet codon specifies one and only one amino acid.

e. The code has start and stop signals: there are one start codon and three stop codons.

10. The Code Is Universal

a. The few exceptions to universality of the genetic code suggest the code dates back to the very first organisms and that all organisms are related.

b. Once the code was established, changes would be disruptive.

12.4 First Step: Transcription

A. Messenger RNA Is Produced

A segment of the DNA helix unwinds and unzips.

Transcription begins when RNA polymerase attaches to a promoter on DNA. A promoter is a region of DNA which defines the start of the gene, the direction of transcription, and the strand to be transcribed.

As RNA polymerase (an enzyme that speeds formation of RNA from a DNA template) moves along the template strand of the DNA, complementary RNA nucleotides are paired with DNA nucleotides of the coding strand. The strand of DNA not being transcribed is called the noncoding strand.

RNA polymerase adds nucleotides to the 3'-end of the polymer under construction. Thus, RNA synthesis is in the 5’-to-3’ direction.

The RNA/DNA association is not as stable as the DNA double helix; therefore, only the newest portion of the RNA molecule associated with RNA polymerase is bound to DNA; the rest dangles off to the side.

Elongation of mRNA continues until RNA polymerase comes to a stop sequence.

The stop sequence causes RNA polymerase to stop transcribing DNA and to release the mRNA transcript.

Many RNA polymerase molecules work to produce mRNA from the same DNA region at the same time.

Cells produce thousands of copies of the same mRNA molecule and many copies of the same protein in a shorter period of time than if a single copy of RNA were used to direct protein synthesis.

B. RNA Molecules Undergo Processing

1. Newly formed pre-mRNA transcript is processed before leaving the nucleus.

2. Pre-mRNA transcript is the immediate product of transcription; it contains exons and introns.

3. The ends of the mRNA molecule are altered: a cap is put on the 5' end and a poly-A tail is put on the 3' end.

a. The “cap” is a modified guanine (G) where a ribosome attaches to begin translation.

b. The “poly-A tail” consists of a 150–200 adenine (A) nucleotide chain that facilitates transport of mRNA out of the nucleus and inhibits enzymatic degradation of mRNA.

4. Portions of the primary mRNA transcript, called introns, are removed.

a. An exon is a portion of the DNA code in the primary mRNA transcript eventually expressed in the final polypeptide product.

An intron is a non-coding segment of DNA removed by spliceosomes before the mRNA leaves the nucleus.

5. Ribozymes are RNAs with an enzymatic function restricted to removing introns from themselves.

a. RNA could have served as both genetic material and as the first enzymes in early life forms.

6. Spliceosomes are complexes that contain several kinds of ribonucleoproteins.

2. The tRNA is a single-stranded ribonucleic acid that doubles back on itself to create regions where complementary bases are hydrogen-bonded to one another.

3. The amino acid binds to the 3’ end; the opposite end of the molecule contains an anticodon that binds to the mRNA codon in a complementary fashion.

4. There is at least one tRNA molecule for each of the 20 amino acids found in proteins.

5. There are fewer tRNAs than codons because some tRNAs pair with more than one codon; if an anticodon contains a U in the third position, it will pair with either an A or G—this is called the wobble hypothesis.

6. The tRNA synthetases are amino acid-activating enzymes that recognize which amino acid should join which tRNA molecule, and covalently joins them. This requires ATP.

7. An amino acid–tRNA complex forms, which then travels to a ribosome to “transfer” its amino acid during protein synthesis.

B. The Role of Ribosomal RNA

1. Ribosomal RNA (rRNA) is produced from a DNA template in the nucleolus of the nucleus.

2. The rRNA is packaged with a variety of proteins into ribosomal subunits, one larger than the other.

3. Subunits move separately through nuclear envelope pores into the cytoplasm where they combine when translation begins.

4. Ribosomes can float free in cytosol or attach to endoplasmic reticulum.